Bottom Line:
In saline group the area of lesion, characterized by lack of glial fibrillary acidic protein immunoreactivity, was of 0.45 ± 0.07 mm(2) in the hippocampal stratum lacunosum-moleculare, and was accompanied by upregulation of laminin immunostaining, and by increased endothelin-1 expression.In addition, JMV-1843 counteracted (P<0.05) the changes in laminin and endothelin-1 expression, both increased in ghrelin-treated rats.These results demonstrate diverse protective effects of growth hormone secretagogues in rats exposed to status epilepticus.

Affiliation: Department of Biomedical, Metabolic and Neural Sciences, University of Modena and Reggio Emilia, Modena, Italy.

ABSTRACTIn models of status epilepticus ghrelin displays neuroprotective effects mediated by the growth hormone secretagogue-receptor 1a (GHS-R1a). This activity may be explained by anticonvulsant properties that, however, are controversial. We further investigated neuroprotection and the effects on seizures by comparing ghrelin with a more effective GHS-R1a agonist, JMV-1843. Rats were treated either with ghrelin, JMV-1843 or saline 10 min before pilocarpine, which was used to induce status epilepticus. Status epilepticus, developed in all rats, was attenuated by diazepam. No differences were observed among the various groups in the characteristics of pilocarpine-induced seizures. In saline group the area of lesion, characterized by lack of glial fibrillary acidic protein immunoreactivity, was of 0.45 ± 0.07 mm(2) in the hippocampal stratum lacunosum-moleculare, and was accompanied by upregulation of laminin immunostaining, and by increased endothelin-1 expression. Both ghrelin (P<0.05) and JMV-1843 (P<0.01) were able to reduce the area of loss in glial fibrillary acidic protein immunostaining. In addition, JMV-1843 counteracted (P<0.05) the changes in laminin and endothelin-1 expression, both increased in ghrelin-treated rats. JMV-1843 was able to ameliorate neuronal survival in the hilus of dentate gyrus and medial entorhinal cortex layer III (P<0.05 vs saline and ghrelin groups). These results demonstrate diverse protective effects of growth hormone secretagogues in rats exposed to status epilepticus.

Mentions:
Adult male Sprague-Dawley rats (Harlan, San Pietro al Natisone, UD, Italy), weighing 230–250 g were used. All the experiments were performed in accordance with the European Directive 2010/63/EU and approved by the Italian Ministry of Health (DM 126/2011 - B). All efforts were done to minimize the number of animals and their sufferance. Pilocarpine (380 mg/kg, i.p.; Sigma-Aldrich, Milan, Italy) was used to induce SE. The pilocarpine injection was preceded by methylscopolamine (1 mg/kg, i.p.) to prevent the peripheral effects of cholinergic stimulation [27]. Ghrelin (1.5 mg/kg; n = 14), JMV-1843 (330 µg/kg; n = 13), or saline (n = 13) were injected i.p. 20 min after scopolamine and 10 min before pilocarpine. Doses were chosen according to our previous experiments [25]. All drugs were dissolved in physiological saline. Diazepam (20 mg/kg, i.p.) was injected 10 min after the SE onset to improve survival and to standardize SE duration. This procedure makes convulsive seizures to stop, leaving non-convulsive seizures unaltered for hours [31]. Behavioral (Table 1) and electrographic (Table 2) seizures were carefully evaluated during the experiment and, subsequently, in video electrocorticography (ECoG) recordings. In non-implanted animals, we considered SE as the stage in which rats did not recover normal behavior (i.e., exploration, grooming or motor reaction to stimuli) between one seizure and the other and, according to this criterion, rats were treated with diazepam approximately 25–38 min after the pilocarpine injection. In implanted rats, we considered SE as the stage where epileptiform electrographic activity was virtually continuous (interruption of high amplitude activity was never longer than 5 s; see Figure 1 bottom traces in each panel), a condition reached approximately 16–18 min after pilocarpine injection (Table 2). Non-epileptic rats (n = 8) received methylscopolamine followed, 30 min later, by saline instead of pilocarpine, but not diazepam, and were used as normal control group used for immunohistochemistry. All rats treated with pilocarpine developed SE and were used for immunohistochemistry (n = 16) or for video ECoG (n = 24).

Mentions:
Adult male Sprague-Dawley rats (Harlan, San Pietro al Natisone, UD, Italy), weighing 230–250 g were used. All the experiments were performed in accordance with the European Directive 2010/63/EU and approved by the Italian Ministry of Health (DM 126/2011 - B). All efforts were done to minimize the number of animals and their sufferance. Pilocarpine (380 mg/kg, i.p.; Sigma-Aldrich, Milan, Italy) was used to induce SE. The pilocarpine injection was preceded by methylscopolamine (1 mg/kg, i.p.) to prevent the peripheral effects of cholinergic stimulation [27]. Ghrelin (1.5 mg/kg; n = 14), JMV-1843 (330 µg/kg; n = 13), or saline (n = 13) were injected i.p. 20 min after scopolamine and 10 min before pilocarpine. Doses were chosen according to our previous experiments [25]. All drugs were dissolved in physiological saline. Diazepam (20 mg/kg, i.p.) was injected 10 min after the SE onset to improve survival and to standardize SE duration. This procedure makes convulsive seizures to stop, leaving non-convulsive seizures unaltered for hours [31]. Behavioral (Table 1) and electrographic (Table 2) seizures were carefully evaluated during the experiment and, subsequently, in video electrocorticography (ECoG) recordings. In non-implanted animals, we considered SE as the stage in which rats did not recover normal behavior (i.e., exploration, grooming or motor reaction to stimuli) between one seizure and the other and, according to this criterion, rats were treated with diazepam approximately 25–38 min after the pilocarpine injection. In implanted rats, we considered SE as the stage where epileptiform electrographic activity was virtually continuous (interruption of high amplitude activity was never longer than 5 s; see Figure 1 bottom traces in each panel), a condition reached approximately 16–18 min after pilocarpine injection (Table 2). Non-epileptic rats (n = 8) received methylscopolamine followed, 30 min later, by saline instead of pilocarpine, but not diazepam, and were used as normal control group used for immunohistochemistry. All rats treated with pilocarpine developed SE and were used for immunohistochemistry (n = 16) or for video ECoG (n = 24).

Bottom Line:
In saline group the area of lesion, characterized by lack of glial fibrillary acidic protein immunoreactivity, was of 0.45 ± 0.07 mm(2) in the hippocampal stratum lacunosum-moleculare, and was accompanied by upregulation of laminin immunostaining, and by increased endothelin-1 expression.In addition, JMV-1843 counteracted (P<0.05) the changes in laminin and endothelin-1 expression, both increased in ghrelin-treated rats.These results demonstrate diverse protective effects of growth hormone secretagogues in rats exposed to status epilepticus.

Affiliation:
Department of Biomedical, Metabolic and Neural Sciences, University of Modena and Reggio Emilia, Modena, Italy.

ABSTRACTIn models of status epilepticus ghrelin displays neuroprotective effects mediated by the growth hormone secretagogue-receptor 1a (GHS-R1a). This activity may be explained by anticonvulsant properties that, however, are controversial. We further investigated neuroprotection and the effects on seizures by comparing ghrelin with a more effective GHS-R1a agonist, JMV-1843. Rats were treated either with ghrelin, JMV-1843 or saline 10 min before pilocarpine, which was used to induce status epilepticus. Status epilepticus, developed in all rats, was attenuated by diazepam. No differences were observed among the various groups in the characteristics of pilocarpine-induced seizures. In saline group the area of lesion, characterized by lack of glial fibrillary acidic protein immunoreactivity, was of 0.45 ± 0.07 mm(2) in the hippocampal stratum lacunosum-moleculare, and was accompanied by upregulation of laminin immunostaining, and by increased endothelin-1 expression. Both ghrelin (P<0.05) and JMV-1843 (P<0.01) were able to reduce the area of loss in glial fibrillary acidic protein immunostaining. In addition, JMV-1843 counteracted (P<0.05) the changes in laminin and endothelin-1 expression, both increased in ghrelin-treated rats. JMV-1843 was able to ameliorate neuronal survival in the hilus of dentate gyrus and medial entorhinal cortex layer III (P<0.05 vs saline and ghrelin groups). These results demonstrate diverse protective effects of growth hormone secretagogues in rats exposed to status epilepticus.